Peripheral arterial disease (PAD) is caused by atherosclerosis that results in obstructions in the arteries to the lower extremities. The prevalence of PAD is nearly equal to that of ischemic heart disease. In a sizeable fraction of patients with severe forms of PAD, there is a complete occlusion in one or more major arteries, and thus blood flow to the distal lower limb becomes dependent on the extent, number and function of collateral blood vessels. There are no medical therapies available for patients with PAD that favorably modulate collateral number or function and perfusion to the lower extremity. The concept of stimulating new blood vessel growth ('therapeutic angiogenesis') to improve limb perfusion in patients with PAD is now a 20 year old investigational strategy. The vast majority of human trials use gene transfer by intra-muscular (IM) delivery with plasmid or adenoviral vectors. After dozens of trials and thousands of subjects, success in this field has been meager. Human studies have failed to convincingly show therapeutic gene expression following IM injection, and this may well account for the failure of clinical trials in therapeutic angiogenesis conducted to date. Adeno-associated virus can be transformational in providing the link from mouse to man for therapeutic angiogenesis in PAD. Following systemic (intravenous) delivery, several AAV serotypes efficiently transduce skeletal muscle. While PAD could potentially limit the access of AAV9 to ischemic muscle, we found enhanced gene expression in ischemic compared to non-ischemic muscle following systemic delivery and we have examined potential mechanisms. Building on this foundation, the central hypothesis of this revised proposal is that bioengineered AAV9 vectors are uniquely suited to accomplish therapeutic angiogenesis in PAD via local delivery using isolated limb perfusion. The three complementary but independent Aims are to: Specific Aim 1: Optimize AAV9-mediated gene delivery to ischemic muscle using systemic vs. isolated limb perfusion in small and large animal models of PAD as a link to clinical application. We will use reporter genes and quantitative biodistribution studies of gene expression to compare systemic versus local delivery in ischemic muscle relative to off-target tissues in mice and pigs. Further, we will incorporate hypoxia-response elements and examine immune responses in both murine and porcine hind-limb ischemia models of PAD. Specific Aim 2: Establish that ischemia-induced desialylation, which results in a difference in the ratio of sialylated to desialylated glycoproteins, is a mechanism for enhanced gene expression in ischemic muscle in pre-clinical and clinical PAD subjects vs. non-PAD controls. Specific Aim 3: Determine the efficacy of AAV-mediated gene therapy delivered via isolated limb perfusion in both mouse and porcine hind-limb ischemia models of PAD. In this aim, we will compare ADAM12 vs. EcSOD gene therapy in the mouse hind-limb ischemia model of PAD, then confirm the efficacy of the most effective gene therapy in the porcine hind-limb model of PAD in collaboration with Dr. Albert Sinusas (Yale).